U.S. patent application number 13/846224 was filed with the patent office on 2013-12-05 for novel thermophilic bacterium and uses of extracellular proteins therefrom.
This patent application is currently assigned to INDUSTRIAL TECHNOLOGY RESEARCH INSTITUTE. The applicant listed for this patent is INDUSTRIAL TECHNOLOGY RESEARCH INSTITUTE. Invention is credited to Jo-Shu CHANG, I-Ju CHOU, Tai-Rong GUO, Yin-Lung HAN.
Application Number | 20130323818 13/846224 |
Document ID | / |
Family ID | 49670709 |
Filed Date | 2013-12-05 |
United States Patent
Application |
20130323818 |
Kind Code |
A1 |
HAN; Yin-Lung ; et
al. |
December 5, 2013 |
NOVEL THERMOPHILIC BACTERIUM AND USES OF EXTRACELLULAR PROTEINS
THEREFROM
Abstract
A novel thermophilic bacterium is provided, which has a 16S rDNA
sequence as set forth in SEQ ID NO. 1. The thermophilic bacterium
excretes extracellular proteins having excellent metal-ion binding
ability, being useful in the treatment of boiler equipment,
pipelines, geothermal wells or industrial wastewater or hard
water.
Inventors: |
HAN; Yin-Lung; (Tainan City,
TW) ; GUO; Tai-Rong; (Zhubei City, TW) ;
CHANG; Jo-Shu; (Taichung City, TW) ; CHOU; I-Ju;
(Tainan City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INDUSTRIAL TECHNOLOGY RESEARCH INSTITUTE |
Hsinchu |
|
TW |
|
|
Assignee: |
INDUSTRIAL TECHNOLOGY RESEARCH
INSTITUTE
Hsinchu
TW
|
Family ID: |
49670709 |
Appl. No.: |
13/846224 |
Filed: |
March 18, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61652522 |
May 29, 2012 |
|
|
|
Current U.S.
Class: |
435/252.1 ;
210/681; 507/244; 530/350 |
Current CPC
Class: |
C12R 1/01 20130101; C09K
8/528 20130101; C07K 14/195 20130101; C02F 5/08 20130101; C12N 1/20
20130101; C12N 1/02 20130101; C23F 11/14 20130101; C02F 2103/10
20130101 |
Class at
Publication: |
435/252.1 ;
507/244; 530/350; 210/681 |
International
Class: |
C12R 1/01 20060101
C12R001/01; C02F 5/08 20060101 C02F005/08; C09K 8/528 20060101
C09K008/528 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 22, 2012 |
TW |
101143616 |
Claims
1. An isolated thermophilic bacterium, Tepidimonas fonticaldi sp.
nov., internationally deposited in the Korean Collection of Type
Culture (KCTC) with deposit number of KCTC 23862.
2. The bacterium of claim 1, comprising a 16S rDNA sequence as set
forth in SEQ ID NO. 1.
3. A protein, excreted extracellularly by the isolated thermophilic
bacterium of claim 1.
4. The protein of claim 3, wherein the protein contains binding
ability to a metal ion(s).
5. The protein of claim 4, wherein the binding is under a
temperature of 75.about.150.degree. C.
6. The protein of claim 4, wherein the binding is under a pressure
of 1.about.50 atm.
7. The protein of claim 4, wherein the binding is under a pH
2.about.10.
8. The protein of claim 4, wherein the metal ion comprises bi- or
tri-valent metal ions.
9. The protein of claim 4, wherein the metal ion comprises cerium
(Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd),
holmium (Ho), lanthanum (La), lutetium (Lu), neodymium (Nd),
praseodymium (Pr), scandium (Sc), samarium (Sm), terbium (Tb),
thorium (Th), thulium (Tm), uranium (U), ytterbium (Yb), yttrium
(Y), silver (Ag), aluminium (Al), boron (B), barium (Ba), bismuth
(Bi), calcium (Ca), cadmium (Cd), cobalt (Co), chromium (Cr),
copper (Cu), iron (Fe), gallium (Ga), indium (In), potassium (K),
lithium (Li), magnesium (Mg), manganese (Mn), sodium (Na), nickel
(Ni), lead (Pb), strontium (Sr), thallium (Tl), zinc (Zn) or
combinations thereof.
10. A method for inhibiting salt formation, comprising contacting
an extracellular protein(s) from an isolated thermophilic bacterium
with a metal ion-containing solution to form a complex of the
protein(s) and the metal ion(s), wherein the isolated thermophilic
bacterium is Tepidimonas fonticaldi sp. nov. internationally
deposited in the Korean Collection of Type Culture (KCTC) with
deposit number of KCTC 23862.
11. The method of claim 10, wherein the isolated thermophilic
bacterium comprises a 16S rDNA sequence as set forth in SEQ ID NO.
1.
12. The method of claim 10, wherein the metal ion comprises bi- or
tri-valent metal ions.
13. The method of claim 10, wherein the metal ion comprises cerium
(Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd),
holmium (Ho), lanthanum (La), lutetium (Lu), neodymium (Nd),
praseodymium (Pr), scandium (Sc), samarium (Sm), terbium (Tb),
thorium (Th), thulium (Tm), uranium (U), ytterbium (Yb), yttrium
(Y), silver (Ag), aluminium (Al), boron (B), barium (Ba), bismuth
(Bi), calcium (Ca), cadmium (Cd), cobalt (Co), chromium (Cr),
copper (Cu), iron (Fe), gallium (Ga), indium (In), potassium (K),
lithium (Li), magnesium (Mg), manganese (Mn), sodium (Na), nickel
(Ni), lead (Pb), strontium (Sr), thallium (Tl), zinc (Zn) or
combinations thereof.
14. The method of claim 10, wherein the method is performed under a
temperature of 75.about.150.degree. C.
15. The method of claim 10, wherein the method is performed under a
pressure of 1.about.50 atm.
16. The method of claim 10, wherein the method is performed under a
pH 2.about.10.
17. The method of claim 10, wherein the metal ion-containing
solution comprises geothermal waters, boiler solutions or
industrial waste or hard water.
18. The method of claim 10, in the treatment of boiler equipment,
water pipelines, geothermal wells or industrial wastewater or hard
water.
19. A method for treating high temperature wastewater, comprising
contacting an extracellular protein(s) from an isolated
thermophilic bacterium with a metal ion-containing solution to form
a complex of the protein(s) and the metal ion(s), wherein the
isolated thermophilic bacterium is Tepidimonas fonticaldi sp. nov.
internationally deposited in the Korean Collection of Type Culture
(KCTC) with deposit number of KCTC 23862.
20. The method of claim 19, wherein the isolated thermophilic
bacterium comprises a 16S rDNA sequence as set forth in SEQ ID NO.
1.
21. The method of claim 19, wherein the metal ion comprises bi- or
tri-valent metal ions.
22. The method of claim 19, wherein the metal ion comprises cerium
(Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium (Gd),
holmium (Ho), lanthanum (La), lutetium (Lu), neodymium (Nd),
praseodymium (Pr), scandium (Sc), samarium (Sm), terbium (Tb),
thorium (Th), thulium (Tm), uranium (U), ytterbium (Yb), yttrium
(Y), silver (Ag), aluminium (Al), boron (B), barium (Ba), bismuth
(Bi), calcium (Ca), cadmium (Cd), cobalt (Co), chromium (Cr),
copper (Cu), iron (Fe), gallium (Ga), indium (In), potassium (K),
lithium (Li), magnesium (Mg), manganese (Mn), sodium (Na), nickel
(Ni), lead (Pb), strontium (Sr), thallium (Tl), zinc (Zn) or
combinations thereof.
23. The method of claim 19, wherein the method is performed under a
temperature of 75.about.150.degree. C.
24. The method of claim 19, wherein the method is performed under a
pressure of 1.about.50 atm.
25. The method of claim 19, wherein the method is performed under a
pH 2.about.10.
26. The method of claim 19, wherein the metal ion-containing
solution comprises geothermal waters, boiler solutions or
industrial waste or hard water.
27. The method of claim 19, in the treatment of boiler equipment,
water pipelines, geothermal wells or industrial wastewater or hard
water.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority of Taiwan Patent
Application No. 101143616, filed Nov. 22, 2012, which claims the
priority of U.S. Provisional Application No. 61/652,522, filed May
29, 2012, the disclosure of which is hereby incorporated by
reference herein in its entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] A sequence listing submitted as a text file via EFS-Web is
incorporated herein by reference. The text file containing the
sequence listing is named "0956-A24070-US_Seq_Listing.txt"; its
date of creation is Mar. 1, 2013; and its size is 2,129 bytes.
TECHNICAL FIELD
[0003] The technical field relates to a novel thermophilic
bacterium and uses of extracellular proteins therefrom.
BACKGROUND
[0004] Geothermal energy is an abundant green energy from the Earth
and has potential for development. However, due to its unique
components, a change of temperature or pressure may easily cause
geothermal fluids to form scales on the wellbore walls or in the
formation fractures, which increases the instability of geothermal
fluid production.
[0005] An acid or chemical treatment has been internationally used
against geothermal scale formation. However, the waste fluids left
from the acid or chemical treatment cause severe environmental
pollution. Therefore, development of green energy with high
production efficiency, while being environmentally friendly, is
being researched more and more.
SUMMARY
[0006] A detailed description is given in the following embodiments
with reference to the accompanying drawings.
[0007] In one embodiment, the disclosure provides an isolated
thermophilic bacterium, Tepidimonas fonticaldi sp. nov.,
internationally deposited in the Korean Collection of Type Culture
(KCTC) with the deposit number of KCTC 23862.
[0008] In another embodiment, the disclosure provides a method for
inhibiting salt formation, which comprises contacting an
extracellular protein(s) from an isolated thermophilic bacterium
with a metal ion-containing solution to form a complex of the
protein and the metal ion. The isolated thermophilic bacterium is
Tepidimonas fonticaldi sp. nov., internationally deposited in the
Korean Collection of Type Culture (KCTC) with the deposit number of
KCTC 23862.
[0009] In another embodiment, the disclosure provides a method for
treating high temperature wastewater, which comprises contacting an
extracellular protein(s) from an isolated thermophilic bacterium
with a metal ion-containing solution to form a complex of the
protein and the metal ion. The isolated thermophilic bacterium is
Tepidimonas fonticaldi sp. nov., internationally deposited in the
Korean Collection of Type Culture (KCTC) with the deposit number of
KCTC 23862.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The present invention can be more fully understood by
reading the subsequent detailed description and examples with
references made to the accompanying drawings, wherein:
[0011] FIG. 1 shows a phylogenic tree diagram of Tepidimonas
fonticaldi sp. nov., based on the similarity of 16S rDNA;
[0012] FIG. 2 is a bar graph showing the Ca.sup.2+ binding ability
of intra- and extra-cellular proteins of several thermophilic
strains;
[0013] FIG. 3 is a bar graph showing the Ca.sup.2+ binding ability
of extracellular proteins of several thermophilic strains under
various temperature conditions;
[0014] FIG. 4 is a bar graph showing the Ca.sup.2+ binding ability
of extracellular proteins of several thermophilic strains under
various pressure conditions;
[0015] FIG. 5 is a bar graph showing the Ca.sup.2+ binding ability
of extracellular proteins of several thermophilic strains under
various pH value conditions;
[0016] FIG. 6 is a bar graph showing the dysprosium (Dy) ion
binding ability of extracellular proteins of several thermophilic
strains;
[0017] FIG. 7 is a bar graph showing the lanthanum (La) ion binding
ability of extracellular proteins of several thermophilic
strains;
[0018] FIG. 8 is a bar graph showing the neodymium (Nd) ion binding
ability of extracellular proteins of several thermophilic
strains;
[0019] FIG. 9 is a bar graph showing the scandium (Sc) ion binding
ability of extracellular proteins of several thermophilic
strains;
[0020] FIG. 10 is a bar graph showing the samarium (Sm) ion binding
ability of extracellular proteins of several thermophilic
strains;
[0021] FIG. 11 is a bar graph showing the ytterbium (Yb) ion
binding ability of extracellular proteins of several thermophilic
strains;
[0022] FIG. 12 is a bar graph showing the barium (Ba) ion binding
ability of extracellular proteins of several thermophilic strains;
and
[0023] FIG. 13 is a bar graph showing the Ca.sup.2+ binding ability
of extracellular proteins of several thermophilic strains.
DETAILED DESCRIPTION
[0024] In the following detailed description, for purposes of
explanation, numerous specific details are set forth in order to
provide a thorough understanding of the disclosed embodiments. It
will be apparent, however, that one or more embodiments may be
practiced without these specific details. In other instances,
well-known structures and devices are schematically shown in order
to simplify the drawing.
[0025] In one embodiment, the present disclosure provides a novel
thermophilic bacterium isolated and purified from hot spring water
at the Antun, Hualien County, Taiwan. The isolated thermophilic
bacterium, after being analyzed and sequenced, shows a sequence of
the 16S ribosomal DNA (rDNA) as set forth in SEQ ID No. 1.
According to the sequence of the 16S rDNA, the isolated strain
shows phylogenetically close to the well-known strains, Tepidimonas
thermarum AA-1.sup.T (97.5% sequence similarity), Tepidimonas
aquatica CLN-1.sup.T (96.8% sequence similarity), Tepidimonas
ignava SPS-1037.sup.T (96.4% similarity) and Tepidimonas
taiwanensis I1.about.1.sup.T (95.8% sequence similarity). A
phylogenetic tree diagram based on the sequence similarity of 16S
rDNA is depicted in FIG. 1.
[0026] The 16S rDNA sequences of the isolated thermophilic strain
and Tepidimonas thermarum AA-1.sup.T were made to hybridize. A
DNA-DNA relatedness was shown as 23.9.+-.0.2%, indicating that the
isolated thermophilic strain belonged to Tepidimonas sp.
[0027] On the biological characteristics, the isolated thermophilic
bacterium shows aerobic and Gram negative, and forms non-pigmented
colonies and mobiles by a single polar flagellum. An optimal growth
occurs at 35.about.60.degree. C. , or 55.degree. C., at 0.about.1.0
wt % of NaCl, or 0.2 wt % of NaCl, and under a pH 7.0.about.9.0, or
pH 7.0. The predominant cellular fatty acids of the isolated
thermophilic strain include C.sub.16:0 (40.2%), summed feature 3
(C.sub.16:1 .omega.7c and/or C.sub.16:1 .omega.6c; 20.1%) and
C.sub.17:0 cyclo (11.5%). The major polar fatty acids include
phosphatidylethanolamine (PE) and phosphatidylglycerole (PG). The
GC content of the total DNA in one cell of the bacterium is 70.1
mol %.
[0028] On the basis of the phylogenetic and phenotypic data, the
isolated strain is classified as a novel species and named as
Tepidimonas fonticaldi sp. nov., internationally deposited in the
Korean Collection of Type Culture (KCTC) on Jan. 10, 2012 with the
deposit number of KCTC 23862. It was also deposited in the
Bioresource Collection and Research Center (BCRC) of Taiwan on Nov.
21, 2011 with the deposit number of BCRC 80391 and was also
deposited in the Laboratorium voor Microbiologie Gent' Belgium
(LMG) on Nov. 28, 2011 with the deposit number of LMG 26746.
[0029] The isolated thermophilic bacterium, Tepidimonas fonticaldi
sp. nov., extracellularly excretes proteins under an appropriate
condition. The extracellular proteins have excellent metal ion
binding ability and are not affected by high temperatures, high
pressure and/or extreme pH conditions. Specifically, in one
example, the extracellular proteins show excellent metal ion
binding ability under a temperature of 75.about.150.degree. C., or
125.about.150.degree. C., at 1 atmosphere (atm), pH 7. In another
example, the extracellular proteins show excellent metal ion
binding ability under a pressure of 1.about.50 atm, or 10.about.30
atm, at 25.degree. C., pH 7. In another example, the extracellular
proteins show excellent metal ion binding ability at pH 2.about.10,
or pH 7.about.10, at 25.degree. C., 1 atm.
[0030] The metal ion binding ability is preferably bindings to bi-
or tri-valent metal ions. The bi- or tri-valent metal ions may
comprise cerium (Ce), dysprosium (Dy), erbium (Er), europium (Eu),
gadolinium (Gd), holmium (Ho), lanthanum (La), lutetium (Lu),
neodymium (Nd), praseodymium (Pr), scandium (Sc), samarium (Sm),
terbium (Tb), thorium (Th), thulium (Tm), uranium (U), ytterbium
(Yb), yttrium (Y), silver (Ag), aluminium (Al), boron (B), barium
(Ba), bismuth (Bi), calcium (Ca), cadmium (Cd), cobalt (Co),
chromium (Cr), copper (Cu), iron (Fe), gallium (Ga), indium (In),
potassium (K), lithium (Li), magnesium (Mg), manganese (Mn), sodium
(Na), nickel (Ni), lead (Pb), strontium (Sr), thallium (Tl), zinc
(Zn) or combinations thereof, but are not limited thereto.
[0031] Due to the excellent metal ion binding ability, the
extracellular proteins are able to compete with acid ions in the
solution for binding with metal ions. Therefore, the metal salt
formed by acid ions binding with metal ions can be reduced so that
the scales formed from the metal salts can be inhibited.
[0032] Accordingly, one embodiment of the present disclosure
provides a method for inhibiting salt formation, comprising
contacting the extracellular protein(s) with a metal ion-containing
solution to form a complex of the protein(s) and the metal
ion(s).
[0033] Another embodiment of the present disclosure provides a
method for treating high temperature wastewater by contacting the
extracellular protein(s) with metal ion-containing wastewater to
form a complex of the protein(s) and the metal ion(s).
[0034] Generally, in high temperature conditions, such as
geothermal waters, boiler solutions or industrial waste or hard
water, acid ions in the water or solutions ordinarily bind to metal
ions to form metal salts, like calcium carbonate (CaCO.sub.3),
which produces scales on the walls of machines, mechanical tubes,
water pipelines or the like, which leads to obstruction or damage.
This causes operation of the machinery to be difficult or even
damaged during high temperatures.
[0035] On the contrary, according to the present invention, the
extracellular proteins is able to prevent scale formation,
especially CaCO.sub.3, on the walls of boiler instruments,
mechanical tubes, water pipelines, geothermal wells, industrial
wastes or hard waters or the like due to its excellent metal ion
binding ability. Therefore, machinery can be well operated in high
temperatures, under high pressure or during extreme pH conditions.
The operation period and cost can be reduced accordingly. In
addition, instead of acid or chemical reagents, the extracellular
proteins of the present disclosure can prevent environmental
pollution from the acidic or chemical treatment. Thus, the
extracellular proteins of the present disclosure are useful in the
treatment of boiler equipment, mechanical tubes, water pipelines,
geothermal wells, industrial wastewater or hard water or the
like.
Example 1
Collection and Identification of Tepidimonas fonticaldi sp.
nov.
[0036] Collection
[0037] 10 L of the hot spring water at the Antun, Hualien County,
Taiwan was collected with an aluminum foil-wrapped bottle. The
collected hot spring water was filtered through a 0.45 .mu.m filter
membrane under a sterile condition. The filter membrane was then
put onto a 1.5% agarose medium (a mixture of 300 mL of hot spring
water and 4.5 g of agarose, under 121.degree. C., heated for 15
minutes and added into a medium plate for cooling) and cultured at
55.degree. C. for 7.about.14 days. After being cultured, colonies
with various colors or shapes were picked up with a sterile
inoculating loop and lined on a fresh agarose medium. The steps
above were repeated to obtain a pure strain. The isolated strain
was stored at 4.degree. C.
[0038] Bacteria DNA Extraction
[0039] The isolated strain was picked up from the medium with a
sterile inoculating loop and added into 1 mL of sterile water.
After three times of being washed with sterile water, the genomic
DNA of the isolated strain was extracted by using a genomic DNA
extraction minoprep system (Blood & Tissue). The extracted
genomic DNA was then stored at -20.degree. C.
[0040] 16S rDNA Sequencing
[0041] The extracted genomic DNA underwent a polymerase chain
reaction (PCR) following the conditions set forth in Tables 1 and 2
with a universal prokaryotic 16S ribosome PCR primer pair of FD1
(5'-AGA GTT TGA TCC TGG CTC AG-3') and RD1 (5'-AAG GAG GTG ATC CAG
CC-3') for amplifying the 16S rDNA sequence of the isolated strain.
The amplified 16S rDNA was sequenced by Mission Biotech
Incorporation, Taiwan.
TABLE-US-00001 TABLE 1 PCR Reagents Volume Reagents (.mu.l) DNA
templates 1 Tag buffer 2.5 (10 X) Tag polymerase (2.5 U/.mu.l)
0.125 dNTP (2.5 mM) 2 Forward primer (100 1 pmol/.mu.l) Reverse
primer (100 1 pmol/.mu.l) deionized water 17.5 Total volume 25
TABLE-US-00002 TABLE 2 PCR Conditions Temperature Cycle and time
Reaction numbers Stage 1 95.degree. C. 5 min Denature 1 Stage 2
95.degree. C. 1 min Denature 35 56.degree. C. 1 min Annealing
72.degree. C. 1 min Extending Stage 3 72.degree. C. 4 min Extending
1
[0042] Alignment of 16S rDNA Sequence
[0043] The 16S rDNA sequence of the isolated strain was aligned
with the GenBank library by using BLAST software
(http://www.ncbi.nlm.nih.gov) and similarities with the published
sequences in the library were analyzed. The 16S DNA sequence of the
isolated strain was recorded in the NCBI (www.ncbi.nlm.nih.gov)
with a series number of JN 713899, named Tepidimonas fonticaldi sp.
nov.
[0044] Construction of the Phylogenic Tree
[0045] According to the sequence similarity obtained above, a
phylogenic tree diagram was depicted with accession numbers of each
strain by using BioEdit software and CLUSTAL_X and homology among
these strains was calculated as well. The phylogenic tree diagram
is shown in FIG. 1, in which the isolated strain was identified as
Tepidimonas fonticaldi sp. nov.
Example 2
Metal Ion Binding Ability of Extracellular Proteins
[0046] Protein Collection
[0047] The isolated strain, Tepidimonas fonticaldi sp. nov., and a
modal strain, Thermus aquaticus BCRC 17110, were respectively
inoculated in a 100 mL 1/10 TSB medium (1.7 g/L pancreatic digest
of casein, 0.3 g/L enzymatic digest of soybean meal, 0.25 g/L
dextrose, 0.5 g/L NaCl and 0.25 g/L dipotassium phosphate) and
cultured at 200 rpm, at 55.degree. C. for 3.about.5 days.
[0048] The cultured medium was centrifuged at 10,000 rpm for 10
minutes. The supernatant was collected and concentrated by 10 times
with a protein concentration system. The concentrated protein was
taken as the extracellular proteins for the next steps. Meanwhile,
the precipitate of the centrifuged medium was ultrasound vibrated
to break the bacteria and the solution was taken as the
intracellular proteins for the next steps.
[0049] Protein Quantification
[0050] The extracellular proteins solution obtained above 100 .mu.L
were added with a Bio-Rad protein test reagent 400 .mu.L for a
color reaction, occurring within 15 minutes. The absorbance at a
wavelength of 595 nm was detected and the protein concentration was
calculated.
[0051] Ca.sup.2+ Binding Ability of Extra- and Intra-Cellular
Proteins
[0052] The experiment was designed for investigating the Ca.sup.2+
binding ability of the extra- and intra-cellular proteins. The
extra- and intra-cellular proteins from the thermophilic bacteria
listed in Table 3 were all detected for the Ca.sup.2+ binding
ability.
[0053] 50 ppm of the extra- and intra-cellular proteins obtained
from the thermophilic bacteria listed in Table 3 were respectively
mixed with the 100 ppm calcium ion solution prepared by
CaCl.sub.2.2H.sub.2O in a ratio of 1:1. The mixture was heated in
100.degree. C. of a water bath for 1 hour. The mixture was then
filtered through an ultrafiltration system with a 3 KDa membrane to
capture the proteins. The filtrate was then diluted with deionized
water to reach the detection limit (0.about.5 ppm). The Ca.sup.2+
concentration left in the filtrate was analyzed by an inductively
coupled plasma atomic emission spectrometer (ICP-AES) for
calculating the Ca.sup.2+ binding ability. The results are shown in
Table 3 and FIG. 2.
TABLE-US-00003 TABLE 3 Ca.sup.2+ Binding Ability of Extra- and
Intra-Cellular Proteins Ca.sup.2+ binding ability (mg Ca.sup.2+/mg
protein) Strain Intracellular Extracellular codes Strains protein
protein IC-5 98.764% of 16 S DNA similarity 0.012 0.034 with
Anoxybacillus kamchatkensis JW/VK-KG4.sup.T LJ-B 98.949% of 16 S
DNA similarity 0.005 0.069 with Anoxybacillus mongoliensis T4.sup.T
AT-A1 100.00% of 16 S DNA similarity 0.006 0.032 with Meiothermus
ruber DSM 1279.sup.T AT-A2 Tepidimonas fonticaldi sp. nov. 0.011
0.327 BCRC Thermus aquaticus BCRC 17110 0.005 0.082 17110
[0054] As shown in Table 3 and FIG. 2, the Ca.sup.2+ binding
ability of the extracellular proteins was much more significant
than that of the intracellular proteins. Meanwhile, the
extracellular proteins of Strain AT-A2 showed 0.327 mg of
Ca.sup.2+/mg protein, the best binding ability among the strains.
Regarding the other strains, the extracellular proteins merely
showed Ca.sup.2+ binding ability of less than 0.1 mg of
Ca.sup.2+/mg protein.
[0055] Ca.sup.2+ Binding Ability Under Various Conditions
[0056] An experiment was designed for investigating the effects of
environmental factors, like temperature, pressure, pH value etc.,
on the Ca.sup.2+ binding ability of the extracellular proteins from
some of the thermophilic bacteria listed in Table 3.
[0057] The experiment included the three following groups:
[0058] Group 1: The extracellular proteins 50 ppm were treated
respectively under a pressure of 10 atm, 30 atm and 50 atm, at
25.degree. C., pH 7 for 10 minutes;
[0059] Group 2: The extracellular proteins 50 ppm were treated
respectively under a pH 2, pH 4, pH 6, pH 7, pH 8 and pH 10 at
25.degree. C., 1 atm for 10 minutes; and
[0060] Group 3: The extracellular proteins 50 ppm were treated
respectively under a temperature of 100.degree. C., 125.degree. C.,
and 150.degree. C., at 1 atm, pH 7 for 10 minutes.
[0061] The treated proteins 50 ppm were separately mixed with a 100
ppm calcium ion solution prepared by CaCl.sub.2.2H.sub.2O in a
ratio of 1:1. The mixture was heated in a 100.degree. C. water bath
for 10 minutes. The mixture was then filtered through an
ultrafiltration system with a 3 KDa membrane to capture the
proteins. The filtrate was then diluted with deionized water to
reach the detection limit (0.about.5 ppm). The Ca.sup.2+
concentration left in the filtrate was analyzed by an inductively
coupled plasma atomic emission spectrometer (ICP-AES) for
calculating the Ca.sup.2+ binding ability. The results are shown in
FIGS. 3-5.
[0062] In FIGS. 3.about.5, the extracellular proteins from Strain
AT-A2 (Tepidimonas fonticaldi sp. nov.) showed excellent Ca.sup.2+
binding ability under high temperatures, high pressure or broad pH
values.
[0063] Bi- and Tri-Valent Metal Ion Binding Ability
[0064] An experiment was designed for investigating the bi- and
tri-valent metal ion binding ability of the extracellular proteins
from some of the thermophilic bacteria listed in Table 3.
[0065] The experiment included the two following groups:
[0066] Group A: The extracellular proteins 10 ppm were mixed with a
first standard solution 10 ppm in a ratio of 1:1 and reacted at
100.degree. C., pH 2 for 20 minutes. The first standard solution
included metal ions of cerium (Ce), dysprosium (Dy), erbium (Er),
europium (Eu), gadolinium (Gd), holmium (Ho), lanthanum (La),
lutetium (Lu), neodymium (Nd), praseodymium (Pr), scandium (Sc),
samarium (Sm), terbium (Tb), thorium (Th), thulium (Tm), uranium
(U), ytterbium (Yb) and yttrium (Y). The binding ability is shown
in a unit of .mu. mole metal ions/mg proteins in FIGS.
6.about.11.
[0067] Group B: The extracellular proteins 10 ppm were mixed with a
second standard solution 10 ppm in a ratio of 1:1 and reacted at
100.degree. C., pH 2 for 20 minutes. The second standard solution
included metal ions of silver (Ag), aluminium (Al), boron (B),
barium (Ba), bismuth (Bi), calcium (Ca), cadmium (Cd), cobalt (Co),
chromium (Cr), copper (Cu), iron (Fe), gallium (Ga), indium (In),
potassium (K), lithium (Li), magnesium (Mg), manganese (Mn), sodium
(Na), nickel (Ni), lead (Pb), strontium (Sr), thallium (Tl) and
zinc (Zn). The Ba.sup.2+ and Ca.sup.2+ binding ability is shown in
a unit of .mu. mole metal ions/mg protein in FIGS. 12.about.13.
[0068] As given in FIGS. 6.about.13, the extracellular proteins
from Strain AT-A2 (Tepidimonas fonticaldi sp. nov.) showed
excellent binding ability broadly to bi- and tri-valent metal
ions.
[0069] It will be apparent to those skilled in the art that various
modifications and variations can be made to the disclosed
embodiments. It is intended that the specification and examples be
considered as exemplary only, with a true scope of the disclosure
being indicated by the following claims and their equivalents.
Sequence CWU 1
1
111276DNATepidimonas thermarum AA-1 1gggtgctgac gagtggcgaa
cgggtgagta atgcatcgga acgtgcccag aggtggggga 60taacgcagcg aaagctgtgc
taataccgca tgtgatctga ggatgaaagc gggggaccaa 120gcagcaatgt
ttggcctcgc gcctctggag cggccgatgt cagattaggt agttggtggg
180gtaaaggcct accaagccga cgatctgtag ctggtctgag aggacgacca
gccacactgg 240gactgagaca cggcccagac tcctacggga ggcagcagtg
gggaattttg gacaatgggc 300gcaagcctga tccagcaatg ccgcgtgcgg
gaagaaggcc ttcgggttgt aaaccgcttt 360tgtacggaac gaaaaggctc
tggctaatac ctggggctga tgacggtacc gtaagaataa 420gcaccggcta
actacgtgcc agcagccgcg gtaatacgta gggtgcgagc gttaatcgga
480attactgggc gtaaagcgtg cgcaggcggt cttgtaagac agaggtgaaa
tccctgggct 540caacctagga atggcctttg tgactgcaag gctggagtgc
ggcagagggg gatagaattc 600cgcgtgtagc agtgaaatgc gtagatatgc
ggaggaacac cgatggcgaa ggcagtcccc 660tgggcctgca ctgacgctca
tgcacgaaag cgtggggagc aaacaggatt agataccctg 720gtagtccacg
ccctaaacga tgtcgactgg ttgttgggcc ttaggtggct cagtaacgaa
780gctaacgcgt gaagtcgacc gcctggggag tacggccgca aggttgaaac
tcaaaggaat 840tgacggggac ccgcacaagc ggtggatgat gtggtttaat
tcgatgcaac gcgaaaaacc 900ttacctaccc ttgacatgcc aggaatcctg
cagagatgtg ggagtgctcg caagagagcc 960tggacacagg tgctgcatgg
ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1020gcaacgagcg
caacccttgc cattagttgc tacgaaaggg cactctaatg ggactgccgg
1080tgacaaaccg gaggaaggtg gggatgacgt caagtcctca tggcccttat
gggtagggct 1140acacacgtca tacaatggcc ggtacagagg gctgccaacc
cgcgaggggg agccaatccc 1200gtaaaaccgg tcgtagtccg gattgcagtc
tgcaactcga ctgcatgaag tcggaatcgc 1260tagtaatcgc ggatca 1276
* * * * *
References